The long-term goal of the proposed work is to determine the role of sequence-specific DNA methylation in the regulation of photoreceptor gene expression. The work is designed to whether DNA regions that regulate gene expression are methylated at specific nucleotide bases in non- photoreceptor cells but unmethylated in photoreceptors. Experiments will be done to determine whether lack of methylation at specific DNA residues permits expression in photoreceptors but methylation prevents expression in other tissues. These questions are consequential because improper gene regulation of photoreceptor- specific proteins can cause retinal degenerations, color vision deficiencies, and quite likely an abundance of other pathologies. This research also is important in that it explores the previously unstudied regulatory role of low-density methylation in photoreceptors, as opposed to high-density methylation, which is relatively well-characterized in non-photoreceptor systems. DNA methylation will be mapped by conversion sequencing, a new technique in which DNA is chemically treated to discriminate methylated from unmethylated nucleotide bases in subsequent sequencing reactions. It is predicted that genes that are selectively expressed in photoreceptors will contain residues that are unmethylated in DNA isolated from photoreceptors but methylated in DNA from other cell types. It is further predicted that if hypomethylation permits gene expression in vivo, then hypomethylation patterns will appear prior to gene transcription initiation. To determine whether these methylation patters regulate gene expression, the effects of exogenous DNA methylation on gene promoter activity will be assessed with transient transfections of cultured retina, retinoblastoma, and non-retina cells. It is predicted that methylation at specific bases identified in mapping experiments will suppress gene promoter activity, thus establishing a causative role for site-specific hypomethylation in photoreceptor gene regulation.